Endothelial Cell Differentiation Protocol

Endothelial cells were differentiated as previously described^{76 (link)} with minor changes. hESCs were plated on Geltrex (ThermoFisher (Gibco), A1413302) coated plates in TeSR-E8TM media (StemCell Technologies, 05990) with 1 μM Rock inhibitor (Y-27632, Selleckchem, S1049). Two days later (d0), cells were collected and reseeded onto non-treated culture dishes coated with 1 μg cm⁻¹ Vitronectin (StemCell Technologies, 07180) at a density of 100,000 cells per cm² in TeSR-E8 medium supplemented with 5 ng ml⁻¹ BMP4 (R&D, 314-BP-050/CF), 25 ng ml⁻¹ Activin A (R&D, 338-AC-050/CF) and 1 μM CHIR99021 (PeproTech, 2520691) for 2 d. At d2, the media were switched to TeSRTM-E7TM media (StemCell Technologies, 05914) supplemented with 50 ng ml⁻¹ VEGF-A (Peprotech, 100-20), 50 ng ml⁻¹ BMP4 (R&D, 314-BP-050/CF) and 5 μM SB431542 (ReproCell, 04-0010-05) for another 4–5 d. Final differentiated cells were stained with antibodies against CD31 (Pe-Cy7; Biolegend,303118) and CD144 (PE; Biolegend, 138009).